cancers Review NRF2 and the Ambiguous Consequences of Its Activation during Initiation and the Subsequent Stages of Tumourigenesis Holly Robertson 1,2, Albena T. Dinkova-Kostova 1 and John D. Hayes 1,* 1 Jacqui Wood Cancer Centre, Division of Cellular Medicine, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, Scotland, UK; [email protected] (H.R.); [email protected] (A.T.D.-K.) 2 Wellcome Trust Sanger Institute, Wellcome Genome Campus, Cambridge CB10 1SA, UK * Correspondence: [email protected]; Tel.: +44-(0)-1382-383182 Received: 30 October 2020; Accepted: 27 November 2020; Published: 2 December 2020 Simple Summary: Transcription factor NRF2 controls expression of antioxidant and detoxification genes. Normally, the activity of NRF2 is tightly controlled in the cell, and is continuously adjusted to ensure that cells are protected against endogenous chemicals and environmental agents that perturb the intracellular antioxidant/pro-oxidant balance (i.e., redox) that must be maintained for them to grow and survive in an appropriate manner. This tight control of NRF2 is achieved by a repressor protein called KEAP1 that perpetually targets NRF2 protein for degradation under normal conditions, but is unable to do so when challenged with oxidants or thiol-reactive chemicals. In the context of cancer, it is well known that drugs that stimulate short-term and reversible activation of NRF2 can provide protection for a limited period against exposure to chemicals that cause cancer. However, it is also becoming widely recognised that permanent hyper-activation of NRF2 resulting from somatic mutations in the gene that encodes NRF2, or in genes associated with its degradation, is frequently observed in certain cancers and associated with poor outcome. In this article, we provide a critical overview of the literature describing the seemingly ambiguous contributions that NRF2 makes to the development of cancer. In particular, we describe the range of genetic and other mechanisms that are responsible for the upregulation of NRF2 in tumours, and highlight shortcomings in our knowledge of how frequently this occurs in different types of cancer. Moreover, we discuss how upregulation of NRF2 might aid the growth and survival of tumours, whether NRF2 upregulation in particular types of cancer is associated with mutations in specific oncogenes, and at what stage of cancer development this is likely to occur. Lastly, we discuss therapeutic strategies that have been proposed that selectively target tumours in which NRF2 is permanently activated with a view to overcoming NRF2-associated drug resistance. Abstract: NF-E2 p45-related factor 2 (NRF2, encoded in the human by NFE2L2) mediates short-term adaptation to thiol-reactive stressors. In normal cells, activation of NRF2 by a thiol-reactive stressor helps prevent, for a limited period of time, the initiation of cancer by chemical carcinogens through induction of genes encoding drug-metabolising enzymes. However, in many tumour types, NRF2 is permanently upregulated. In such cases, its overexpressed target genes support the promotion and progression of cancer by suppressing oxidative stress, because they constitutively increase the capacity to scavenge reactive oxygen species (ROS), and they support cell proliferation by increasing ribonucleotide synthesis, serine biosynthesis and autophagy. Herein, we describe cancer chemoprevention and the discovery of the essential role played by NRF2 in orchestrating protection against chemical carcinogenesis. We similarly describe the discoveries of somatic mutations in NFE2L2 and the gene encoding the principal NRF2 repressor, Kelch-like ECH-associated protein 1 (KEAP1) along with that encoding a component of the E3 ubiquitin-ligase complex Cullin 3 (CUL3), which result in permanent activation of NRF2, and the recognition that such mutations occur frequently in many types Cancers 2020, 12, 3609; doi:10.3390/cancers12123609 www.mdpi.com/journal/cancers Cancers 2020, 12, 3609 2 of 48 of cancer. Notably, mutations in NFE2L2, KEAP1 and CUL3 that cause persistent upregulation of NRF2 often co-exist with mutations that activate KRAS and the PI3K-PKB/Akt pathway, suggesting NRF2 supports growth of tumours in which KRAS or PKB/Akt are hyperactive. Besides somatic mutations, NRF2 activation in human tumours can occur by other means, such as alternative splicing that results in a NRF2 protein which lacks the KEAP1-binding domain or overexpression of other KEAP1-binding partners that compete with NRF2. Lastly, as NRF2 upregulation is associated with resistance to cancer chemotherapy and radiotherapy, we describe strategies that might be employed to suppress growth and overcome drug resistance in tumours with overactive NRF2. Keywords: NRF2; KEAP1; Cullin 3; ATF4; oxidative stress; reactive oxygen species; antioxidant; adaptation; glutathione; thioredoxin; NADPH generation; pentose phosphate pathway; proteasome; autophagy; drug metabolism; chemoprevention; chemotherapy; bioactivation; quinone-containing drugs; drug resistance; oncogene; tumour suppressor; initiation; progression; metastasis; recurrent disease; lung; oesophagus; liver; head and neck; stomach; bladder; colon; rectum 1. Introduction The cap’n’collar (CNC) basic-region leucine zipper (bZIP) transcription factor NF-E2 p45-related factor 2 (NRF2, encoded by NFE2L2) is a master regulator of intracellular redox homeostasis because, in response to oxidative stress, it orchestrates induction of a battery of genes that serve to increase the antioxidant capacity of the cell. Since oxidative stress is associated with many common chronic debilitating ailments such as cancer, cardiovascular disease, diabetes mellitus, inflammatory disease, liver cirrhosis, lung fibrosis and neurodegenerative disease, it is not surprising that pharmacological agents that reversibly activate NRF2, and so alleviate oxidative stress, have been linked to the prevention or attenuation of many of these conditions [1]. Paradoxically, however, in certain types of cancer the irreversible genetic upregulation of NRF2 resulting from stochastic somatic activating mutations in NFE2L2 or inactivating mutations in the gene encoding the repressor of NRF2, Kelch-like ECH-associated protein 1 (KEAP1), or that for its E3 ligase Cullin 3 (CUL3), is associated with progression of disease once it has been initiated [2]. In mammals, oxidative stress increases with age and this is accompanied by increased susceptibility to degenerative disease, which is associated with lower levels of intracellular antioxidants and the downregulation of NRF2 [3–5]. Considerable interest therefore exists around the interplay between degenerative disease, oxidative stress and NRF2, and how this can be exploited to improve health. Although NRF2 was initially discovered by Yuet Wai Kan and colleagues as a human transcription factor that shared homology with the p45 subunit of NF-E2 and was capable of binding a tandemly-arrayed activator protein-1 (AP-1) recognition sequence (i.e., 50-TGAGTCATGATGAGTCA-30, with AP-1-binding sites underlined) in the β-globin gene locus, its involvement in directing cellular adaptation to oxidative stress was not immediately apparent [6,7]. This is possibly because many transcription factors, including AP-1, Forkhead box O (FOXO), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), nuclear factor-kappaB (NF-κB) and tumour protein p53 (TP53), control expression of antioxidant genes, and because NRF2 does not regulate directly the classic antioxidant enzyme superoxide dismutase (SOD) 1 or SOD2, unlike some of these other transcription factors [8]. Rather than being linked to the oxidative stress response, it was thought by various researchers that since the DNA sequence bound by NRF2 resembles an antioxidant responsive element (ARE, 50-TGACNNNGC-30)[9], also called an electrophile responsive element [10], the transcription factor might mediate induction of genes for drug-metabolising enzymes. Of particular significance, the DNA binding site also resembles a musculoaponeurotic fibrosarcoma (MAF) recognition element G (MARE, 50-TGCTGA /CTCAGCA-30)[11], and so the family of small MAF bZIP proteins, which can heterodimerize with NRF2, were considered as possible partners of NRF2 [12]. Cancers 2020, 12, x 3 of 48 Cancers 2020, 12, 3609 3 of 48 [11], and so the family of small MAF bZIP proteins, which can heterodimerize with NRF2, were considered as possible partners of NRF2 [12]. Based on aa mousemouse genegene knockoutknockout (ko) (ko) model, model, the the first first physiological physiological role role of of Nrf2 Nrf2 was was reported reported by Masiby Masi Yamamoto Yamamoto and and colleagues colleagues to be to that be ofthat mediating of mediating induction induction of drug-metabolising of drug-metabolising enzymes, enzymes, in liver andin liver small and intestine, small intestine, by the phenolic by the phenolic antioxidant antioxidant butylated butylated hydroxyanisole hydroxyanisole (BHA), the (BHA), genes the of whichgenes wereof which known were to known contain to ARE contain sequences ARE sequences in their regulatory in their regulatory regions; these regions; included these genesincluded encoding genes NAD(P)H:quinoneencoding NAD(P)H:quinone oxidoreductase oxidoreductase 1 (NQO1, also1 (NQO1, called NAD(P)Halso called dehydrogenase NAD(P)H dehydrogenase (quinone 1), and(quinone occasionally 1), and occasionally DT-diaphorase DT-diaphorase or menadione or menadione reductase)